The present invention relates to a novel hardly water-soluble hyaluronic acid gel composition comprising hyaluronic acid and a polymer, which is substantially unmodified with a chemical cross-linking agent or a chemical modifying agent, and a process for its production. Further, it relates particularly to a medical material utilizing it and having good biocompatibility.
Hyaluronic acid is a linear macromolecular polysaccharide consisting of alternately bonded xcex2-D-N-acetylglucosamine and xcex2-D-glucuronic acid, and it is known to have no species or organ specificity and show excellent biocompatibility even when implanted or injected into the body.
Various chemical modifications of hyaluronic acid have been proposed in order to overcome drawbacks of hyaluronic acid in its application in vivo as a medical material, such that it is easily soluble in water and its in vivo residence time is relatively short. Further, many hyaluronic acid compositions have been studied for modification by an addition of a polymer to supplement various physical properties required for a medical material such as the strength and tissue bondability.
For example, in order to use a hyaluronic acid composition as a bone-repairing material, a higher strength is required as compared with a case where it is used for an artificial cartilage for a joint. Likewise, when a hyaluronic acid composition is used as an adhesion preventive, a higher tissue bondability is required as compared with a case where it is used as an embolizing agent.
Heretofore, a hyaluronic acid composition useful as a medical material may, for example, be a hyaluronic acid composition having sodium hyaluronate and carboxymethyl cellulose, modified with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) as a carbodiimide, as reported in e.g. JP-A-5-508161 and JP-A-6-508169. Further, W086/00912 reports on a hyaluronic acid composition having a carboxy-containing polysaccharide (such as sodium hyaluronate, carboxymethyl dextran, carboxymethyl starch or carboxymethyl cellulose) cross-linked with e.g. BDDE (1,4-butanediol glycine ether) as a di- or poly-functional epoxide. Further, JP-A-61-164558 reports on a hyaluronic acid composition having sodium hyaluronate, chondroitin sulfate, heparin or the like cross-linked with a polyfunctional epoxide, or with cyan bromide, epichlorohydrin or the like. Further, JP-A-61-138601 reports on a hyaluronic acid composition having sodium hyaluronic acid or various polymers cross-linked with divinylsulfone. Further, JP-A-6-73102 and JP-A-8-301903 report on a hyaluronic acid composition having sodium hyaluronate or various polymers cross-linked with cinnamic acid.
However, in their production, it is very difficult to completely remove the cross-linking agent, etc. for purification. Not only that, hyaluronic acid and polymers are likely to include such a cross-linking compound in the molecules, and their physiological functions, biocompatibility and safety can not hardly be said to be substantially equivalent to hyaluronic acid and the polymers.
A hyaluronic acid composition which employs no chemical cross-linking agent or no chemical modifying agent in order to maximize the excellent characteristics of biocompatibility inherent to hyaluronic acid and the polymer itself and which is useful as a biocompatible medical material and has a long in vivo residence time, has not yet been developed.
In order to accomplish the above object, the present inventors have strenuously studied the physiochemical properties of a hyaluronic acid gel (PCT/JP98/03536) itself employing no chemical cross-linking agent or no chemical modifying agent, which is composed solely of hyaluronic acid and which is excellent in biocompatibility and moldability and has an in vivo degradable nature.
Further, they have found that a hyaluronic acid gel composition comprising hyaluronic acid and a polymer, which is substantially unmodified with a chemical cross-linking agent or a chemical modifying agent, will supplement the inherent properties of a hyaluronic acid gel such as strength, adhesiveness, viscosity and elasticity and also will satisfy the physical properties required for a medical material which can hardly be satisfied by a hyaluronic acid gel alone, and it can simply be prepared and has ideal biocompatibility and retention property as a medical material, and they have arrived at the present invention.
Especially when carboxymethyl cellulose is employed as the polymer of this hyaluronic acid gel composition, the composition is particularly suitable as an adhesion preventive or a wound dressing.
Namely, the present invention is (1) a hyaluronic acid gel composition comprising hyaluronic acid and a polymer, which is substantially unmodified with a chemical cross-linking agent or a chemical modifying agent and which has a percentage dissolution of hyaluronic acid of 50% or lower in 12 hours in a neutral aqueous solution of 37xc2x0 C., (2) the hyaluronic acid gel composition according to (1), wherein the polymer is carboxymethyl cellulose, (3) a process for producing a hyaluronic acid gel composition, which comprises freezing an aqueous solution or dispersion containing hyaluronic acid and a polymer and having a pH of 3.5 or lower, followed by unfreezing to form a hyaluronic acid gel composition, (4) a process for producing a hyaluronic acid gel composition, which comprises freezing an aqueous solution containing hyaluronic acid and having a pH of 3.5 or lower, followed by unfreezing to form a hyaluronic acid gel, and mixing the hyaluronic acid gel with a polymer or a polymer gel to form a hyaluronic acid gel composition, (5) the process for producing a hyaluronic acid gel composition according to (3) or (4), wherein the polymer is at least one member selected from the group consisting of polysaccharides, proteins, nucleic acids and synthetic polymers, (6) the process for producing a hyaluronic acid gel composition according to (3) or (4), wherein the polymer is carboxymethyl cellulose, (7) a medical material containing the hyaluronic acid gel composition as defined in (1) or (2), (8) a medical material containing a hyaluronic acid gel composition which is obtained by irradiating or injecting one member selected from the group consisting of gamma rays, electron rays, plasma or EOG to a hyaluronic acid gel composition, (9) the medical material according to (7), wherein the material is an adhesion preventive, and (10) the medical material according to (7), wherein the medical material is a wound dressing.
Now, the present invention will be described in detail.
In the present invention, modification means cross-linking or chemical modification to make naturally water-soluble hyaluronic acid or polymer to be hardly soluble.
The hyaluronic acid gel composition of the present invention can be obtained by freezing an aqueous solution or dispersion having a pH of 3.5 or lower containing hyaluronic acid and a polymer, followed by unfreezing and by neutralization, washing and drying.
Further, the hyaluronic acid gel composition of the present invention can be obtained by freezing an aqueous solution containing hyaluronic acid and having a pH of 3.5 or lower, followed by unfreezing to obtain a hyaluronic acid gel, which is then pulverized and put, together with a polymer gel, into an aqueous solution such as water, followed by pulverization, dispersion and drying.
With these hyaluronic acid gel compositions, the mechanical strength and the tissue bondability can simply be improved as compared with a case of the hyaluronic acid gel alone.
Any acid may be used as the acid to be employed to adjust the pH of the aqueous solution of hyaluronic acid and the polymer, so long as it is an acid capable of adjusting the pH to a level of 3.5 or lower. In order to reduce the amount of the acid, it is preferred to use a strong acid such as hydrochloric acid, nitric acid or sulfuric acid.
For freezing and unfreezing, an operation wherein a acidic aqueous solution having hyaluronic acid and a polymer adjusted, is put into an optional container and then frozen at a predetermined temperature, and after completion of the freezing, unfreezing is carried out at a predetermined temperature, at least once. The temperatures and the times for freezing and unfreezing may suitably be determined within ranges of the temperatures and the times for freezing and unfreezing of the acidic aqueous solution of hyaluronic acid, depending upon the size of the container and the amount of the aqueous solution. However, usually, a freezing temperature of lower than the freezing point and an unfreezing temperature of higher than the freezing point, are preferred. Since the freezing and unfreezing times can be shortened, a freezing temperature of xe2x88x925xc2x0 C. or lower and an unfreezing temperature of 5xc2x0 C. or higher are selected more preferably. Further, the times are not particularly limited so long as they are sufficient to complete the freezing and the unfreezing at the respective temperatures.
The number of repetitions of the operation of freezing the acidic aqueous solution having hyaluronic acid and the polymer adjusted, followed by unfreezing, is suitably determined depending upon the molecular weight of hyaluronic acid to be used, the concentration of the aqueous solution, the pH of the aqueous solution, the temperatures and times of the freezing and unfreezing, and various properties such as strength of the hyaluronic acid gel composition to be formed. Usually, it is preferred to repeat the operation at least once.
Every time when the operation of freezing and unfreezing is repeated, the temperatures and the times for the freezing and unfreezing may be changed.
With respect to treatment such as molding of the hyaluronic acid gel composition, at the time of preparation, by selection of a method or a container for freezing the acidic solution having hyaluronic acid adjusted, it is possible to prepare a hyaluronic acid gel composition in a desired form such as a sheet-form, a film-form, a pulverized form, a sponge-form, a bulk form, a fiber-form, a fluid-form or a tube-form.
For example, a hyaluronic acid gel composition of a sheet-form can be obtained by putting the hyaluronic acid composition or its dispersion in a container having a flat bottom, followed by freeze drying.
Further, a hyaluronic acid gel composition of a film-form can be obtained by putting the hyaluronic acid composition or its dispersion in a container having a flat bottom, followed by drying in air.
Hyaluronic acid to be used in the present invention may be one extracted from an animal tissue, or one produced by a fermentation method, and it may be used irrespective of its origin.
The molecular weight of hyaluronic acid to be used in the present invention is preferably one within a range of from about 1xc3x97105 to about 1xc3x97107 Da. Further, so long as it is one having a molecular weight within the above range, even one obtained from one having a higher molecular weight by e.g. hydrolytic treatment, may likewise preferably be used.
Further, in the present invention, hyaluronic acid is used in a concept which includes also an alkali metal salt such as a sodium, potassium or lithium salt.
As the polymer to be used in the present invention, it is possible to use any polymer which is capable of forming a hyaluronic acid gel composition of the present invention irrespective of a natural polymer or a synthetic polymer, and any polymer which is capable of supplementing the inherent properties of the hyaluronic acid gel for the physical properties required for a medical material which can hardly be satisfied by the hyaluronic acid gel alone.
Accordingly, all polymers which will be taken into a hyaluronic acid gel without being involved in formation of cross-linking among polymers themselves or between a polymer and a hyaluronic acid gel and which are capable of forming a hyaluronic acid gel composition of the present invention, can be used for the present invention.
Typical examples of the polymer to be used in the present invention are selected from the group consisting of polysaccharides, proteins, nucleic acids and synthetic polymers, but the useful polymer is not limited thereto.
Examples of polysaccharides may be glycosaminoglycans (such as heparin, heparan sulfate and dermatan sulfate), chondroitin sulfates (such as chondroitin-6-sulfate), keratin sulfates, heparan sulfates, alginic acid and its biologically acceptable salts, cellulose, chitin, chitosan, dextran, starch, amylose, polyactic acid and calaginan.
Further, synthetic derivatives of polysaccharides may, for example, be carboxymethyl cellulose, carboxymethyl amylose, various alkyl celluloses, hydroxyethyl cellulose, carboxy cellulose and oxidized starch and oxidized regenerated cellulose.
Further, examples of proteins may be collagen, gelatin, albumin, elastin, various globulins, casein, gluten, and their biologically acceptable synthetic derivatives.
Further, examples of the synthetic polymer may be polyvinyl alcohol, polyethylene glycol, polyglycolic acid, polyacrylic acid, polymethacrylic acid, copolymers thereof, and derivatives such as poly(hydroxyethyl)acrylates or methacrylates, polyacrylamides, polyvinyl alcohols and copolymers of maleic acid or fumaric acid.
Further, the present invention is by no means restricted to these polymers.
The aqueous solution or dispersion containing hyaluronic acid and a polymer and having a pH of 3.5 or lower, to be used in the present invention, can be obtained by mixing and stirring hyaluronic acid and a polymer with water. The concentrations of hyaluronic acid and the polymer are preferably 5.0 mass % or lower, respectively, from the viewpoint of handling efficiency of the aqueous solution or dispersion.
Especially when hyaluronic acid having a molecular weight of 2xc3x97106 Da or higher, is used, the concentration of the hyaluronic acid is preferably 2.5 mass % or lower. The blend ratio of the aqueous solution or dispersion containing hyaluronic acid and a polymer and having a pH of 3.5 or lower, is not particularly limited, so long as a hyaluronic acid gel composition can be obtained by freezing and unfreezing this liquid. For example, the blend ratio as an adhesion preventive, is preferably from 50:1 to 1:20.
Further, in the present invention, a hyaluronic acid gel and a polymer gel may be prepared separately, and they may be pulverized to obtain a hyaluronic acid gel pulverized product and a polymer gel pulverized product, and such pulverized products may be mixed to obtain a hyaluronic acid gel composition.
The ratio of mixing the hyaluronic acid gel and the polymer gel to be used in the present invention is not particularly limited so long as the mixture can be put into an aqueous solution such as water and can be pulverized and dispersed.
The hyaluronic acid gel composition obtained by the present invention may be used without any particular restriction as a general biodegradable biomedical material in any fields wherein hyaluronic acid is used. It may be used for, e.g. an adhesion preventive, an artificial cartilage for a joint, a carrier for a pharmacologically active substance, a wound dressing, an artificial skin, a replacement vital tissue repairer, a joint injection, a surgical suture, a hemostatic material, an artificial organ, an artificial extracellular matrix, an artificial basement membrane or biomedical products such as medical tools and devices for diagnostic or therapeutic use or medicinal compositions. Further, it is possible to obtain a hyaluronic acid gel composition containing a physiologically active substance by mixing the physiologically active substance when the hyaluronic acid gel and the polymer gel are mixed.
Then, a medical material obtained by irradiation treatment of the hyaluronic acid gel composition of the present invention, will be described. The xcex3-rays to be irradiated to the hyaluronic acid gel composition are preferably produced from cobalt 60 or cesium 137 as a radiation source. When xcex3-rays having a dose of 30 kGy or lower are preferably irradiated to a dried product of the hyaluronic acid gel composition, a hyaluronic acid gel composition effective for an adhesion preventive or a wound treating agent, can be obtained. Further, by changing the irradiation dose or the irradiation time, the solubility of the hyaluronic acid gel composition can be controlled, and it is possible to control a proper in vivo retention property in an application as a biological material. A sterilization effect is also expected by irradiation of xcex3-rays to the hyaluronic acid gel composition.
Electron rays to be irradiated to the hyaluronic acid gel composition are generated by an electron ray accelerator. When electron rays having a dose of 30 kGy or lower are preferably irradiated to a dry product of the hyaluronic acid gel composition, a hyaluronic acid gel composition effective for an adhesion preventive or a wound dressing, can be obtained.
Plasma is represented as the fourth state of substance distinguishable from solid, liquid and gas, and it usually comprises ions, electrons and neutral nuclear species. The gas ionized by applying an electric power to the gas stream, is known also as glow discharge.
As the plasma to be irradiated to the hyaluronic acid gel composition, a low temperature gas plasma to be formed by exposing a mixture of hydrogen, oxygen and an inert carrier gas to an electromagnetic field, may, for example, be used.
A dried product of the hyaluronic acid gel composition is put in a chamber of a plasma generator, and a plasma-generating gas comprising argon, oxygen and hydrogen, is injected and dispersed and then irradiated in a plasma atmosphere for at least 10 minutes, whereby a hyaluronic acid gel composition effective for an adhesion preventive or a wound dressing, can be obtained.
EOG is usually a sterilization method employing ethylene oxide gas, which is applied to a material which can not be subjected to dry sterilization or vapor sterilization. A dried product of the hyaluronic acid gel composition is put into an EOG sterilization chamber, EOG is then injected, and sterilization is carried out preferably at a temperature of 50xc2x0 C. or lower, such being effective for an adhesion preventive or a wound dressing.
By changing the temperature or the time for injecting EOG, the solubility of the hyaluronic acid gel composition can be controlled, and it is possible to control a proper in vivo retention property in its application as a biological material.
Now, an adhesion preventive among medical materials of the present invention will be described.
The adhesion preventive made of a hyaluronic acid gel composition obtained according to the present invention is a sheet-like, filmy, flaky, spongy, massive, fibrous, fluid-form or tubular material for surgical use. With respect to the mode of use, it is preferred to directly apply a filmy or sheet-like material to a part subjected to surgery. It is also preferred to apply a fine flaky or fluid-form material by injection to a part subjected to surgery. It is also useful for peritoneoscopical surgery.
Further, an adhesion preventive made of a hyaluronic acid gel composition encapsulating a physiologically active compound can be obtained by mixing a prepared acidic hyaluronic acid gel composition solution and a physiologically active compound and then freezing and unfreezing the mixture.
The adhesion preventive made of a hyaluronic acid gel composition obtained according to the present invention may be administered at any time during or after the operation so long as postoperative adhesion can be prevented, but preferably immediately before the completion of the operation.